The effects of two cytotoxic gold(i) carbene compounds on the metabolism of A2780 ovarian cancer cells: mechanistic inferences through NMR analysis

NMR metabolomics is a powerful tool to characterise the changes in cancer cell metabolism elicited by anticancer drugs. Here, the large metabolic alterations produced by two cytotoxic gold carbene compounds in A2780 ovarian cancer cells are described and discussed in comparison to auranofin, in the frame of the available mechanistic knowledge.

8  Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2023
-Au(NHC)Cl and [Au(NHC) 2 ]PF 6 were previously prepared and characterized as described in ref 1.; their purity and homogeneity were again checked prior to use by elemental analysis and 1 H NMR analysis.
A2780 cells were seeded in 60 cm 2 tissue-culture plates at 2.5 × 10 5 cells/ mL (total volume 8 mL) and incubated for 6, 12 and 24 h, then exposed to concentrations of AF, Au(NHC)Cl or

NMR experiments
The one-dimensional (1D) 1 H NMR spectra were recorded with a Bruker 600 MHz spectrometer (5,6), (Bruker BioSpin) optimized for metabolomic analysis, operating at 600.13 MHz proton Larmor frequency and equipped with a 5 mm PATXI 1 H-13 C-15 N and 2 Hdecoupling probe including a z-axis gradient coil, an automatic tuning-matching (ATM) and an automatic refrigerated sample changer (SampleJet, Bruker BioSpin). A BTO 2000 thermocouple served for temperature stabilization at the level of approximately 0.1 K of the sample. Before measurement, to equilibrate temperature at 300 K, samples were kept for at least 5 minutes inside the NMR probe head.
The cell lysate samples were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) sequence using a one-dimensional (1D) spin-echo sequence with water presaturation; 512 scans, 73728 data points, a spectral width of 12019 Hz and a relaxation delay of 4 s were used ( Figure S1).
The growth media were acquired with a 1D nuclear Overhauser enhancement spectroscopy (NOESY)-presaturation pulse sequence; 64 scans, 98304 data points, a spectral width of 18028 Hz and a relaxation delay of 4 s were used ( Figure S1).
The raw data were multiplied by a 0.5 Hz exponential line broadening before applying Fourier transform. Transformed spectra were automatically corrected for phase and baseline distortions and calibrated at the doublet of Ala at 1.49 ppm using TopSpin 3.6 (Bruker Biospin srl).

Data analysis
The metabolites, whose peaks in the spectra were well resolved, were assigned and their levels analyzed using an in-house developed R script. We could identify and quantify 30 metabolites in the cell lysate spectra and 28 in the culture medium spectra (endo-and exo-metabolome in Figure. S1, respectively). The assignment was performed using an internal 1 H NMR spectral library of pure organic compounds (BBIOREFCODE, Bruker BioSpin srl), Chenomx software, public databases such as the Human Metabolome Database, stored reference NMR spectra of metabolites and literature data. Matching between new NMR data and databases was performed using the AMIX software (Bruker BioSpin srl). To further confirm the assignment of the most abundant and significant cell lysate metabolites and remove ambiguities, we used the natural abundance 1 H-13 C HSQC spectrum ( Figure S2) and a spiking approach ( Figure S3). The relative concentrations of the various metabolites were calculated by integrating the corresponding signals in the spectra using a home-made R script.
The nonparametric pairwise Wilcoxon-Mann-Whitney test was used to determine the meaningful metabolites; a p-value <0.05 was considered statistically significant. In order to reduce false discoveries, false discovery rate correction (FDR) was then applied using the Benjamini and Hochberg method. Log 2 Fold change (FC) was calculated for each metabolite to display how the metabolite levels vary upon the different comparison. FC is calculated as the median of the ratio of the metabolite concentrations in the spectra of the two paired samples (treated vs. control). In the growth media, metabolites were divided into two different classes, i.e., those that are taken up from the medium and those that are released into the medium. For the molecules that are released, lower/higher concentration levels upon treatment correspond to a lower/higher release, while for the molecules that are taken up from the growth media, lower levels upon treatment correspond to a greater consumption of nutrients, i.e., increased uptake, and viceversa for higher levels.
PCA analysis was performed on binned NMR spectra, using R softaware. To this aim, each spectrum in the region 10.00-0.2 ppm was divided into 0.02 ppm chemical shift bins, and the corresponding spectral areas were integrated using the AMIX software. The area of each bin was normalized to the total spectral area, calculated with exclusion of the water and DMSO region (4.50-5.00 ppm and 2.90-2.60 ppm, respectively).